The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that the prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.
Polymerase Chain Reaction (PCR) is a technique involving multiple cycles that results in the exponential amplification of certain polynucleotide sequences each time a cycle is completed. The technique of PCR is well known and is described in many books, including, PCR: A Practical Approach M. J. McPherson, et al., IRL Press (1991), PCR Protocols: A Guide to Methods and Applications by Innis, et al., Academic Press (1990), and PCR Technology: Principals and Applications for DNA Amplification H. A. Erlich, Stockton Press (1989). PCR is also described in many US patents, including U.S. Pat. Nos. 4,683,195; 4,683,202; 4,800,159; 4,965,188; 4,889,818; 5,075,216; 5,079,352; 5,104,792; 5,023,171; 5,091,310; and 5,066,584.
The PCR technique typically involves the step of denaturing a polynucleotide, followed by the step of annealing at least a pair of primer oligonucleotides to the denatured polynucleotide, i.e., hybridizing the primer to the denatured polynucleotide template. After the annealing step, an enzyme with polymerase activity catalyzes synthesis of a new polynucleotide strand that incorporates the primer oligonucleotide and uses the original denatured polynucleotide as a synthesis template. This series of steps (denaturation, primer annealing, and primer extension) constitutes a PCR cycle.
As cycles are repeated, the amount of newly synthesized polynucleotide increases exponentially because the newly synthesized polynucleotides from an earlier cycle can serve as templates for synthesis in subsequent cycles. Primer oligonucleotides are typically selected in pairs that can anneal to opposite strands of a given double-stranded polynucleotide sequence so that the region between the two annealing sites is amplified.
Denaturation of DNA typically takes place at around 90 to 95° C., annealing a primer to the denatured DNA is typically performed at around 40 to 60° C., and the step of extending the annealed primers with a polymerase is typically performed at around 70 to 75° C. Therefore, during a PCR cycle the temperature of the reaction mixture must be varied, and varied many times during a multicycle PCR experiment.
The PCR technique has a wide variety of biological applications, including for example, DNA sequence analysis, probe generation, cloning of nucleic acid sequences, site-directed mutagenesis, detection of genetic mutations, diagnoses of viral infections, molecular “fingerprinting” and the monitoring of contaminating microorganisms in biological fluids and other sources.
In addition to PCR, other in vitro amplification procedures, including ligase chain reaction as disclosed in U.S. Pat. No. 4,988,617 to Landegren and Hood, have also been developed. More generally, several important methods known in the biotechnology arts, such as nucleic acid hybridization and sequencing, are dependent upon changing the temperature of solutions containing sample molecules in a controlled fashion.
Conventional techniques including PCR typically rely on the use of individual wells or tubes cycled through different temperature zones. For example, a number of thermal “cyclers” used for DNA amplification and sequencing reactions have been described in which a temperature controlled element or “block” holds a reaction mixture, and wherein the temperature of the block is varied over time. Whilst a relatively large number of samples can be processed simultaneously with such devices (e.g. 96 well plates are commonly employed), such devices suffer various drawbacks, in that they are relatively slow in cycling the reaction mixtures, temperature control is less than ideal and detection of the reaction mixture in situ is difficult. Also, such blocks only allow for a “batch” mode of operation since the entire block is simultaneously cooled and heated. Further, it will be appreciated that since it is relatively inconvenient to operate the block if there are only a few samples to run, operators of such devices typically prefer to wait until they have sufficient samples to occupy the majority of the available sample positions, meaning that any urgent samples must wait. Further still, if the temperature cycling routine is started, any vacant positions typically cannot be utilized until the thermal cycling routine is complete and any urgent samples must again wait until the cycle is complete.
In an effort to avoid several of these disadvantages, recent advances have seen the development of block thermal cyclers providing the simultaneous running of different temperature profiles, e.g. as disclosed in U.S. Pat. No. 5,601,141 and U.S. Pat. No. 6,558,947. However, these thermocyclers suffer their own drawbacks. For example, they are relatively expensive and complex, require constant routine maintenance, temperature control is less than ideal and detection of the reaction(s) occurring the reaction containers is still difficult.
Thus, there still remains a need for thermocyclers for PCR which provide accurate temperature control of the reaction mixtures, are not complex to use, can provide real-time analysis of the reaction occurring in the sample containers, and which can operate in a batch or a continuous mode whereby samples can be continuously added and/or removed from the thermocycler during cycling without thermally affecting samples which have only received part of the required number of thermal cycles.
The present invention seeks to overcome or ameliorate at least one of the disadvantages of the abovementioned arrangements, or to provide an alternative to existing arrangements.